Cooperative Research Centre for Tropical Plant Pathology, The University of Queensland, Brisbane, 4072, Australia

Background and objectives
Phytophthora sojae (syn. Phytophthora megasperma f. sp. glycinea) causes a destructive root and stem rot of soybean (Glycine max). Thirteen dominant resistance genes exist in soybean. Eleven corresponding single avirulence genes have been demonstrated to exist in P. sojae [1, 2]. Avirulence is dominant in nine Avr/Rps interactions (Avr1b, 1d, 1k, 3a, 3b, 3c, 4, 5, 6), recessive in one instance (Avr1c), and dependent on the particular parents used in the cross in one instance (Avr1a). Ten of these avirulence genes have been placed on the P. sojae genetic linkage map [2]. RAPD markers have been identified tightly linked to the co-segregating dominant avirulence genes Avr4 and Avr6. The aim of the present research is to use chromosome-walking techniques to clone these two co-segregating avirulence genes.

Materials and methods
The cosmid vector SuperCos 1 (Stratagene) was used to clone P. sojae DNA fragments up to 45 kbp in length. The RAPD marker nearest to Avr4/6 was used as a probe to isolate the first cosmid for chromosome walking. Subsequent cosmids were isolated by using the ends of the first cosmid as probes. The direction of the walk was determined by using the isolated cosmids as RFLP probes to the F2 population. Three contiguous cosmids, thought to span the Avr4/6 region, were identified. Gene expression on contiguous cosmids in the region of Avr4/6 was examined by Northern blot analysis of total P. sojae RNA isolated from mycelium grown under a range of different nutritional conditions. A cDNA library was constructed from P. sojae mRNA exhibiting the greatest diversity of expressed genes. The library was screened with the three contiguous cosmids to isolate positively hybridizing clones.

Results and conclusions
Six RAPD and one RFLP marker were identified in an 18.5 cM region surrounding Avr4/6. The nearest flanking markers to Avr4/6 were 2 and 8.3 cM distant. A chromosome walk was commenced from the nearest marker. A series of three overlapping cosmids were identified which span the genomic region containing Avr4/6. The contiguous cosmids span approximately 70 kbp which represents 12.1 cM.

Northern analysis of total P. sojae RNA revealed low levels of expression for at least three genes. Two genes are expressed constitutively and one is expressed in the absence of an organic nitrogen source.

Using the cosmids containing Avr4/6 as a probe to the cDNA library, a total of 35 cDNA clones representing nine gene sequences of three size classes were isolated. Sequence analysis and comparisons with available databases are currently in progress. Transformation of P. sojae races virulent on Rps4 and Rps6 with cloned cDNA will be carried out to determine whether any function as avirulence genes. For this purpose a highly expressed promotor is being isolated from P. sojae. Cloned avirulence genes may be useful in understanding the interaction between an Oomycete root pathogen and the host plant soybean which is expected to have evolved separately from comparable interactions in the higher fungi. Ultimately, understanding the mechanism of recognition in the host plant will lead to more durable deployment of resistance genes.

1. BM Tyler, H Forster, MD Coffey, 1995. Molecular Plant-Microbe Interactions 8, 515-23.
2. SC Whisson, A Drenth, DJ Maclean, JAG Irwin, 1995. Molecular Plant-Microbe Interactions 8, 988-995.